instaictions): PROJECT 2 - Evidence supports a role for urinary bladder urothelial cells in the transduction of sensory events. Though studies have focussed on the effects of spinal cord injury (SCI) on innervation and smooth muscle, little is known regarding changes in urothelial barrier and signaling properties. We have identified a number of abnormalities in the urothelium (UT) of SCI bladders including alterations in barrier function and a hyperplasia as well as a heightened sensitivity to mechanical and chemical stimuli. Changes to the urothelial barrier occur as early as a few hours following SCI and may be mediated in part by an interaction with bladder nerves (directly or indirectly via urothelial-cell interactions). In contrast, the regenerative process (occurs within the first few weeks following SCI) takes place when bladder nerves are quiescent, suggesting a role for additional factors (including trophic factors). These processes are not mutually exclusive, but interact in a complex manner culminating in altered UT structure and function that can impact on bladder function. The goals of this project are to evaluate and understand mechanisms that underiie both the eariy and delayed changes in the UT that occur following SCI. Aim #1 will evaluate the mechanisms by which neurotransmitters promote acute barrier disruption by measuring transepithelial resistance and permeability. We will also examine whether pelvic nerve stimulation mimics the acute changes of SCI on UT barrier function. Aim #2 will evaluate mechanisms underlying delayed responses of the UT following SCI. We will evaluate whether chronic overdistension of the bladder plays a role in urothelial structure and proliferative function and the involvement of NGF by use of chronic antibody treatment in SCI animals. Aim #3 will continue to examine the impact of chronic SCI on UT function by use of in-line isolated cells, bladder sheets and cross-sections of intact bladder and optical imaging to examine transmitters that may influence UT-cell communication. The Ca2+ sensing protein, GCaMP4, will also be used to measure afferent-UT dynamics in a physiologically relevant context. This use of different experimental approaches will help us elucidate mechanisms underlying changes in cellular responses in SCI. RELEVANCE (See instructions): The bladder urothelium, in addition to its barrier function, plays an important role in the transduction of sensory events. Understanding how the urothelial barrier is disrupted by SCI then repaired and how changes in epithelial function impacts on sensory signaling is of major physiological importance (potentially providing therapeutic targets to alleviate the downstream effects of nerve injuries).